This application claims priority to German Patent Application No. 19910044.6 filed Mar. 8, 1999.
The present invention relates to viral particles which are released after HCMV infection of mammalian cells, and to their use as vaccine.
Human cytomegalovirus (HCMV), a xcex2-herpesvirus is a ubiquitously occurring pathogen. In an immunocompetent person, HCMV infection is normally unnoticed, having at the most mild and nonspecific symptoms. By contrast, in certain risk groups, for example in immunosuppressed patients such as AIDS patients or transplant recipients, and after prenatal infection, HCMV infection has serious manifestations.
Chemotherapeutics are available for treating HCMV infections. The success of antiviral chemotherapy of HCMV infection is restricted, however, in particular by the toxicity of the medicaments and the development of resistant variants of the virus if the duration of treatment is prolonged. In addition, the prophylactic or therapeutic use of antiviral hyperimmune sera has proved to be of only limited efficacy.
There has been work on the development of a vaccine against HCMV for many years. Thus, attempts have been made with weakened (attenuated) live vaccines to induce the desired immunity. This vaccine proved to have only limited efficacy, however. The reasons for this may be, inter alia, the restricted viability of such attenuated viruses in humans and strain-specific variations in the antigenicity. Besides the inadequacies in the induction of a permanent immunity, the use of a live vaccine must be regarded critically; lack of knowledge about the pathogenetic mechanisms in HCMV infection and the risk of reactivating the vaccine strain after immunosuppression make the use of a live vaccine appear at least questionable in these clinical situations.
In order to avoid these risks, strategies have recently been preferentially followed to develop subunit vaccines against HCMV which contain proteins from the viral envelope synthesized in various expression systems. Such envelope proteins, especially the glycoproteins gB and gH, are the essential target antigens of neutralizing antibodies against HCMV. Neutralizing antibodies are able to prevent the infection. It was possible both in experimental animals and in clinical studies to induce such neutralizing antibodies with a gB subunit vaccine. However, in humans, the antibody response induced in this way proved to be short-lived and not suitable for preventing the infection in all cases. This is detrimental to the wide use of subunit vaccines based exclusively on the gB of HCMV. The reasons which have been suggested for the limited efficacy of such antigen preparations in turn are the strain-specific variations in the immune response, lack of induction of an adequate cellular immune response, and structural restrictions of the antigen used, whose epitopes are in some cases known to be conformation-dependent.
On the basis of this experience, therefore, the requirements to be met by an effective and widely useful vaccine against HCMV are as follows:
(1) Long-lasting induction of neutralizing antibodies which protect from HCMV infection in a strain-overlapping manner. This requires efficient induction of a so-called xe2x80x9chelper cell responsexe2x80x9d (CD4-positive T lymphocytes) against HCMV to assist the maturation of antibody-secreting B lymphocytes.
(2) Induction of the formation of cytotoxic T cells against HCMV. Lymphocytes of this type are of crucial importance for terminating an HCMV infection which has taken place and limiting the spread of the virus in the body.
(3) Minimizing the side effects by the vaccine. The risk which might derive from an inoculated viable virus which, according to present knowledge, would have the ability to establish latency after immunosuppression cannot be estimated. The aim ought therefore to be to prepare nonviable viral antigen as vaccine.
In order to comply with the conditions mentioned for an HCMV vaccine, the vaccine must contain the relevant antigens for inducing neutralizing antibodies and for stimulating helper cells (TH lymphocytes) and cytotoxic T cells (CTL).
Neutralizing antibodies are, according to the present state of knowledge, after HCMV infection formed exclusively against viral envelope proteins, and especially against the glycoproteins gB and gH.
TH cells are formed mainly against tegument proteins of the virus, and particularly against the so-called pp65 (ppUL83). In addition, pp65 is an essential antigen for the induction of CTL against HCMV. Presentation of pp65 takes place not only as usual after de novo synthesis by cells in connection with MHC class I molecules; it can also be introduced into the MHC I presentation pathway by so-called xe2x80x9cexogenous loadingxe2x80x9d.
Said antigens are the essential constituents of defective viral particles which, during the infection of primary human fibroblast cultures, are synthesized by HCMV and released into the culture medium. These so-called dense bodies (DB) are structures which are visible under the electron microscope and more than 90% of whose protein mass consists of pp65. They are comparable with virus particles in being provided with a cellular lipid membrane modified by viral glycoproteins and being ejected from the cell. The viral glycoproteins are very probably in the natural conformation in this envelope. Since DB contain no viral DNA and no viral capsid, they are non-infectious. They can be concentrated in large quantity from the cell culture supernatant by established methods.
One object of the invention was to provide an effective and widely usable vaccine against HCMV.
The present invention describes viral particles which are released after infection of mammalian cells by human cytomegalovirus (HCMV). These particles can be employed as preventive or therapeutic vaccine against infections by HCMV.
The particles of the invention are surrounded by a lipid membrane which makes it possible to fuse the particles to certain mammalian cells so that their contents enter the cytoplasm of the cells. The membrane of the particles contains viral glycoproteins which represent the main antigens for virus-neutralizing antibodies. The particles are also characterized in that they contain no viral DNA and no capsid. In addition, they contain the viral T-cell antigen pp65 (ppUL83) which both stimulates the formation of T-helper cells and is an essential antigen for inducing cytotoxic T lymphocytes (CTL) against HCMV.
These properties, especially the combination of antigens able to induce both neutralizing antibodies and an adequate cellular response, make the particles suitable as vaccines against HCMV.
Dense bodies have already been described in the literature (Gibson et al. Birth Defects: Original Article Series 20, 1 (1984) 305-324; Virology 66 (1975) 464-473). In this connection, their possible use as vaccine was suggested. However, no experiments proving that dense bodies in fact show the hoped-for effects were shown. In addition, it has been shown that dense bodies have an antigenic effect (Jahn et al., J. gen. Virol. 68 (1987) 1327-1337). However, these experiments do not characterize the induced immune response, which is why no information about the suitability as vaccine can be inferred.
The fact that the particles of the invention have a high antigenicity and are able to induce the formation of neutralizing antibodies is shown in examples 1 and 2. The induction of virus-neutralizing antibodies is long-lasting (example 3), which is another prerequisite for a successful vaccine. The immune response achieved in examples 1-3 is all the more surprising since the immunization was carried out without using adjuvant. Side effects of vaccines administered with adjuvants thus do not apply. The particles of the invention also activate cytotoxic T lymphocytes (CTL) (examples 4 and 5).
Finally, DB induce, irrespective of the route of administration, T-helper cell responses of the Th1 type (example 6).
These examples demonstrate that the particles of the invention are suitable as vaccine against HCMV.
In a further embodiment, particles which contain a fusion protein which comprises in one part one or more sections of the viral T-cell antigen pp65 (ppUL83) or the complete protein and in another part one or more sections of one or more other proteins are described.
This makes it possible to optimize the antigenicity of the particles because this fusion protein is present in large quantity in the particles. It is additionally known that expression of antigens of the cellular and humoral immune response in one molecule can distinctly increase the antigenicity. The various sections of pp65 and the other proteins can be fused together directly but it is also possible for example for linker sequences, which are not a natural constituent of one of the proteins involved, to be present between the various sections. Sequences of this type may arise because of the cloning or be introduced deliberately in order to influence the properties of the antigen. However, the fusion protein preferably contains no foreign sequences which are not a constituent of one of the fusion partners. In such embodiments, the fusion protein consists of one or more parts of pp65 and one or more parts of one or more other proteins.
It applies to all the embodiments mentioned hereinafter that the complete pp65 or one or more parts thereof can be present in the fusion protein. The statement xe2x80x9ca fusion protein (consisting) of pp65xe2x80x9d is not for the purposes of this application to be understood as restricted to complete pp65. A xe2x80x9cpartxe2x80x9d or xe2x80x9csectionxe2x80x9d of a protein present in the fusion protein comprises at least 6, preferably at least 8, most preferably at least 9, 15 or 20 consecutive amino acids of the protein from which it is derived.
A preferred embodiment comprises a fusion protein of pp65 (ppUL83) and one or more neutralizing epitopes of the viral glycoproteins gB or gH. Particles of this type can be generated as depicted in FIG. 7A. The fusion protein can enter, via antigen-specific uptake, glycoprotein-specific B cells which in turn are able to present the epitopes both of the glycoproteins and of pp65 in the context of MHC class II. In addition, it is also possible for portions of the fusion protein to be presented by professional antigen-presenting cells (APC) in the context of MHC class II. In both cases the result is efficient stimulation of the TH response both to the pp65 and to viral glycoproteins. These TH cells are able to stimulate glycoprotein-specific B cells, which present peptides of pp65 and viral glycoproteins in the context of MHC class II, to form neutralizing antibodies both homologously and heterologously. In addition, particles of this type can, like infectious virions, be taken up into cells, and peptides of pp65 can be introduced by exogenous loading into the MHC class I pathway. This achieves, unusually for dead vaccines, a stimulation of the CTL response to HCMV.
In a further preferred embodiment, the particles contain a fusion protein consisting of pp65 and one or more parts of another protein of HCMV, the IE1 protein (ppUL123). The parts of the IE1 protein which are to be present in particular are those against which cytotoxic T cells are formed in humans during natural infection. Peptides of the IE1 protein are in some cases presented by different MHC class I molecules than are peptides of pp65. The addition of such further xe2x80x9cCTL epitopesxe2x80x9d from IE1 is intended to ensure that, after immunization, inoculated subjects who express different MHC class I molecules are able to generate CTL against HCMV in as comprehensive a manner as possible.
In a further preferred embodiment, the particles contain a fusion protein consisting of pp65, of one or more neutralizing epitopes of HCMV glycoproteins and of one or more CTL epitopes of IE1. Fusion of pp65 with neutralizing epitopes and CTL epitopes is intended to ensure that it is possible simultaneously for both neutralizing antibodies and CTL to be formed by inoculated subjects in as comprehensive a manner as possible, i.e. by the maximum number of people differing in MHC class I pattern.
In a further preferred embodiment, the particles contain a fusion protein of pp65 and one or more epitopes of another human pathogen. Suitable portions of other human pathogens are antigens against which neutralizing antibodies are formed in humans. It is possible through fusion of such xe2x80x9cneutralizing antigensxe2x80x9d with the T-cell antigen pp65 to expect a marked increase in the immune response (antibody response) compared with the use of the isolated xe2x80x9cneutralizing antigenxe2x80x9d. Examples of such xe2x80x9cneutralizing antigensxe2x80x9d which should be mentioned are surface proteins of hepatitis B virus (from the HBsAG region), of hepatitis C virus (for example E2), of human immunodeficiency viruses (HIV, from the Env region), of influenza virus (hemagglutinin, neuraminidase, nucleoprotein) or other viral pathogens. Further suitable human pathogens are bacteria such as Haemophilus influenzae, Bordetella pertussis, Mycobacterium tuberculosis, Neisseria meningitidis and others. Finally, antigens from eukaryotic pathogens such as plasmodia (malaria) could be fused to pp65.
In a further preferred embodiment, the particles contain a fusion protein consisting of pp65 and one or more portions of proteins of other pathogens against which CTL are generated in humans on natural infection with these pathogens. Examples of such CTL epitopes which may be mentioned are portions of proteins of HIV-1, of HBV, of HCV or of influenza virus. The intention of such a procedure is to utilize the unique immunogenic properties of DB for generating protective CTL against heterologous pathogens in humans.
In a further preferred embodiment, the particles contain a fusion protein consisting of pp65, of one or more neutralizing epitopes of a heterologous pathogen and of one or more CTL epitopes of the same pathogen. This fusion is intended to ensure that inoculated subjects are able to form both protective antibodies and CTL against this pathogen.
The invention additionally relates to viral particles containing at least 2 different glycoproteins which are variants of the same glycoprotein from different HCMV strains.
A preferred embodiment contains exactly 2 variants, one variant corresponding to the HCMV Towne strain, and the other variant corresponding to the HCMV Ad169 strain. The preferred embodiment contains the glycoprotein gB both of the Towne strain and of the Ad169 strain.
These two proteins can be incorporated with identical efficiency into the membrane of recombinant dense bodies in the infected cell. Such recombinant dense bodies are suitable for inducing not only the strain-overlapping but also the strain-specific neutralizing immune response to the two prototype HCMV strains.
Finally, the invention further relates to a method by which viral particles which are completely free of infectious virus particles are prepared. If particles are produced from a cell population which has been infected with HCMV there is a risk that infectious virus particles will be carried along during the purification of the particles. This represents a disadvantage for a vaccine.
The method of the invention minimizes this risk. To this end, initially an HCMV strain harboring a deletion in an essential gene is produced. By this is meant a deletion of the function of the gene. In most cases, this is based on the absence of a functional gene product, but it is also possible for the function of a regulatory gene sequence to be deranged in such a way that the HCMV is no longer viable. This can take place by altering the nucleic acid sequence of HCMV, for example by point mutations, actual deletions, insertions or other mutations. This defective virus can replicate only in cells which express the gene which has been deleted in HCMV and thus make it available for assembly of the virions. Primary fibroblasts at present represent the only reasonably permissive system for the in vitro replication of HCMV. Stable transfection of such cells has to date been possible only with the aid of retroviral transfer methods. This is, however, a serious disadvantage if such cells are to be used to produce vaccines. The method of the invention makes available stably transfected cells which can be produced without retroviral gene transfer but in which HCMV can also be replicated.
A preferred embodiment comprises human foreskin fibroblasts which have been stably transfected with the major capsid protein gene UL86. The transfection is preferably carried out with a lipid-containing aid which leads to a very high transfection efficiency. In a preferred embodiment, the xe2x80x9cFugene reagentxe2x80x9d which can be purchased from Roche Diagnostics, Mannheim, is employed for the transfection. This embodiment is described in example 7.
Defective virus whose major capsid protein gene UL86 has been deleted can be replicated in these cells. If xe2x80x9cnon-complementingxe2x80x9d fibroblasts are infected with this defective virus, it is then possible to isolate therefrom viral vaccine particles free of infectious virus particles.
Another possibility for producing the particles of the invention without a risk of infection is to reconstitute the particles in cells without infecting with HCMV. To this end, all the genes which code for constituents of the particles must be expressed in these cells. These genes must for this purpose be inserted into the cells.
Insect cells infected by baculoviruses are preferably used for this purpose. The genes which code for the polypeptide constituents of the particles are cloned into baculovirus expression vectors. The production of recombinant baculoviruses is followed by coinfection of insect cells, preferably Sf9 cells, by the various viruses. The genes are expressed in the insect cells, and the resulting polypeptides combine to give the desired particles. Finally, the particles are released by the insect cells. This represents one possibility for producing noninfectious particles which can be used as vaccine.
An alternative possibility is to clone the constituents necessary for reconstitution of DB into recombinant baculoviruses under the control of the HCMV major IE promoter/enhancer (MIEP). It is shown that recombinant baculoviruses are able to infect higher eukaryotic cells such as, for example, mammalian cells, and that foreign genes under the control of a strong eukaryotic promoter such as MIEP are strongly expressed in such cells. The advantage of such a procedure would be that any important modifications, such as glycosylation, of antigenic proteins of the DB could take place in a more natural manner in mammalian cells than in insect cells. In addition, there is a number of such cell lines which are already approved for vaccine production.